U.S. patent number 6,060,458 [Application Number 09/023,726] was granted by the patent office on 2000-05-09 for oligodeoxyribonucleotides comprising o.sup.6 -benzylguanine and their use.
This patent grant is currently assigned to Arch Development Corporation, The Penn State Research Foundation, The United States of America as represented by the Department of Health. Invention is credited to M. Eileen Dolan, Robert C. Moschel, Gary T. Pauly, Anthony E. Pegg.
United States Patent |
6,060,458 |
Moschel , et al. |
May 9, 2000 |
Oligodeoxyribonucleotides comprising O.sup.6 -benzylguanine and
their use
Abstract
The present invention provides a single-stranded
oligodeoxyribonucleotide, which (i) comprises from about 5 to 11
bases, at least one of which is a substituted or an unsubstituted
O.sup.6 -benzylguanine, and (ii) inactivates human AGT. The present
invention also provides a single-stranded oligodeoxyribonucleotide,
which can inactivate a mutant human AGT, which either is not
inactivated by O.sup.6 -benzylguanine or is less inactivated by
O.sup.6 -benzylguanine than by said single-stranded
oligodeoxyribonucleotide. A phosphate of the single-stranded
oligodeoxyribonucleotide can be replaced by a methylphosphonate or
a phosphorothioate. The present invention also provides a
composition comprising such an oligodeoxyribonucleotide. In
addition, the present invention provides a method of enhancing the
effect of an antineoplastic alkylating agent, which alkylates the
O.sup.6 position of guanine residues in DNA, in the
chemotherapeutic treatment of cancer in a mammal, which method
comprises the co-administration to the mammal of a cancer-treatment
effective amount of an antineoplastic alkylating agent and a
chemotherapeutic treatment-enhancing amount of a present inventive
oligodeoxyribonucleotide or composition thereof.
Inventors: |
Moschel; Robert C. (Frederick,
MD), Pauly; Gary T. (Frederick, MD), Pegg; Anthony E.
(Hershey, PA), Dolan; M. Eileen (Oak Park, IL) |
Assignee: |
The United States of America as
represented by the Department of Health (Washington, DC)
The Penn State Research Foundation (University Park, PA)
Arch Development Corporation (Chicago, IL)
|
Family
ID: |
21816845 |
Appl.
No.: |
09/023,726 |
Filed: |
February 13, 1998 |
Current U.S.
Class: |
514/44A; 435/375;
536/23.1 |
Current CPC
Class: |
A61P
43/00 (20180101); A61P 35/00 (20180101); C07H
21/00 (20130101) |
Current International
Class: |
C07H
21/00 (20060101); A61K 031/70 (); C07H 021/04 ();
C12N 005/06 () |
Field of
Search: |
;435/6,15,193,366,375
;514/44,45
;536/23.1,24.3,24.31,24.32,24.33,24.5,25.3,25.32,27.81 |
References Cited
[Referenced By]
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4199574 |
April 1980 |
Schaeffer |
4495190 |
January 1985 |
Hagberg et al. |
4751221 |
June 1988 |
Watanabe et al. |
4801710 |
January 1989 |
MacCoss et al. |
4965270 |
October 1990 |
Harnden et al. |
5075445 |
December 1991 |
Jarvest et al. |
5091430 |
February 1992 |
Moschel et al. |
5352669 |
October 1994 |
Moschel et al. |
5358952 |
October 1994 |
Moschel et al. |
5364904 |
November 1994 |
Farmer et al. |
5525606 |
June 1996 |
Moschel et al. |
5691307 |
November 1997 |
Moschel et al. |
|
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184473 |
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Aug 1986 |
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335355 |
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EP |
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WO 91/13898 |
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WO |
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WO 94/29312 |
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Dec 1994 |
|
WO |
|
WO 96/04281 |
|
Feb 1996 |
|
WO |
|
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|
Primary Examiner: Elliott; George C.
Assistant Examiner: Larson; Thomas G
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.
Claims
What is claimed is:
1. A single-stranded oligodeoxyribonucleotide of not less than 5
and not more than 11 bases in length comprising at least one
substituted or unsubstituted O.sup.6 -benzylguanine, wherein said
single-stranded oligodeoxyribonucleotide inactivates human
alkyltransferase.
2. The single-stranded oligodeoxyribonucleotide of claim 1, wherein
said oligodeoxyribonucleotide is of not less than 7 bases in
length.
3. The single-stranded oligodeoxyribonucleotide of claim 2, wherein
said oligodeoxyribonucleotide is of not less than 9 bases in
length.
4. The single-stranded oligodeoxyribonucleotide of claim 1, wherein
said O.sup.6 -benzylguanine is substituted with from one to five
substituents, which can be the same or different and are selected
from the group consisting of hydro, halo, haloalkyl, hydroxy,
hydroxyamino, hydrazino, an alkyl, an aryl, nitro, a polycyclic
aromatic alkyl, a cycloalkyl, an alkenyl, an alkynyl, a
hydroxyalkyl, an alkoxy, an alkoxyalkyl, an aryloxy, an acyloxy, an
acyloxyalkyl, a monoalkylamino, a dialkylamino, an acylamino, a
ureido, a thioureido, a carboxy, a carboxyalkyl, a cyano, a
cyanoalkyl, C-formyl, C-acyl, a dialkoxyalkyl, and SO.sub.n
R.sub.1, wherein n is an integer from zero to three and R.sub.1 is
hydro, a C.sub.1 -C.sub.6 alkyl, or a C.sub.1 -C.sub.4
alkyl-substituted or an unsubstituted aryl.
5. The single-stranded oligodeoxyribonucleotide of claim 4, wherein
said from one to five substituents are further substituted.
6. The single-stranded oligodeoxyribonucleotide of claim 4, wherein
said haloalkyl is a C.sub.2 -C.sub.6 straight-chain or a C.sub.3
-C.sub.6 branched-chain alkyl, which is substituted with from one
to three halo groups, said alkyl is a C.sub.2 -C.sub.6
straight-chain or a C.sub.3 -C.sub.6 branched-chain alkyl, said
aryl is substituted with a C.sub.1 -C.sub.8 straight-chain or a
C.sub.3 -C.sub.8 branched-chain alkyl, said polycyclic aromatic
alkyl comprises from two to four aromatic rings and a C.sub.1
-C.sub.6 straight-chain or a C.sub.3 -C.sub.6 branched-chain alkyl,
said cycloalkyl is a C.sub.3 -C.sub.8 cycloalkyl, said alkenyl is a
C.sub.2 -C.sub.6 straight-chain or a C.sub.4 -C.sub.6
branched-chain alkenyl, said alkynyl is a C.sub.2 -C.sub.6
straight-chain or a C.sub.4 -C.sub.6 branched-chain alkynyl, said
hydroxyalkyl is a C.sub.1 -C.sub.6 straight-chain or a C.sub.3
-C.sub.6 branched-chain hydroxyalkyl, said alkoxy is a C.sub.1
-C.sub.8 straight-chain or a C.sub.3 -C.sub.8 branched-chain
alkoxy, said alkoxyalkyl is a C.sub.2 -C.sub.8 alkoxyalkyl, said
acyloxyalkyl comprises a C.sub.1 -C.sub.6 straight-chain or a
C.sub.3 -C.sub.6 branched-chain alkyl, said monoalkylamino and said
dialkylamino comprise a C.sub.1 -C.sub.6 straight-chain or a
C.sub.3 -C.sub.6 branched-chain alkyl, said carboxyalkyl comprises
a C.sub.1 -C.sub.6 straight-chain or a C.sub.3 -C.sub.6
branched-chain alkyl, said cyanoalkyl comprises a C.sub.1 -C.sub.6
straight-chain or a C.sub.3 -C.sub.6 branched-chain alkyl, and said
dialkoxyalkyl comprises a C.sub.1 -C.sub.6 straight-chain or a
C.sub.3 -C.sub.6 branched-chain alkoxy, which can be the same or
different, and a C.sub.1 -C.sub.6 straight-chain or a C.sub.3
-C.sub.6 branched-chain alkyl.
7. The single-stranded oligodeoxyribonucleotide of claim 1, wherein
said at least one substituted or unsubstituted O.sup.6
-benzylguanine is flanked by an equal number of bases in the
3'-direction as in the 5'-direction.
8. The single-stranded oligodeoxyribonucleotide of claim 1, in
which at least one phosphate is modified.
9. The single-stranded oligodeoxyribonucleotide of claim 8, wherein
said at least one phosphate is replaced by a methylphosphonate or a
phosphorothioate.
10. The single-stranded oligodeoxyribonucleotide of claim 8,
wherein said at least one phosphate is a terminal internucleoside
phosphate.
11. The single-stranded oligodeoxyribonucleotide of claim 10,
wherein two terminal internucleoside phosphates are independently
replaced.
12. The single-stranded oligodeoxyribonucleotide of claim 1, which
also inhibits a mutant human alkyltransferase, which either is not
inactivated by O.sub.6 -benzylguanine or is less inactivated by
O.sup.6 -benzylguanine than by said single-stranded
oligodeoxyribonucleotide.
13. The single-stranded oligodeoxyribonucleotide of claim 1,
wherein said oligodeoxyribonucleotide is selected from the group
consisting of
5'-d (GAb.sup.6 GCT)-3', 5'-d (TGAb.sup.6 GCTG)-3', 5'-d
(GTGAb.sup.6 GCTGT)-3', and
5'-d (TGTGAb.sup.6 GCTGTG)-3'. [SEQ ID NO: 1].
14. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 1 and a pharmaceutically
acceptable carrier.
15. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 2 and a pharmaceutically
acceptable carrier.
16. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 3 and a pharmaceutically
acceptable carrier.
17. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 4 and a pharmaceutically
acceptable carrier.
18. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 5 and a pharmaceutically
acceptable carrier.
19. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 6 and a pharmaceutically
acceptable carrier.
20. A composition comprising the single-stranded
oligodeoxyribonucleotide
of claim 7 and a pharmaceutically acceptable carrier.
21. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 8 and a pharmaceutically
acceptable carrier.
22. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 9 and a pharmaceutically
acceptable carrier.
23. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 10 and a pharmaceutically
acceptable carrier.
24. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 11 and a pharmaceutically
acceptable carrier.
25. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 12 and a pharmaceutically
acceptable carrier.
26. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 13 and a pharmaceutically
acceptable carrier.
27. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 1.
28. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 2.
29. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 3.
30. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 4.
31. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 5.
32. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 6.
33. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 7.
34. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 8.
35. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 9.
36. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 10.
37. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 11.
38. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 12.
39. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 13.
40. A single stranded oligodeoxyribonucleotide that inactivates
human alkyltransferase comprising the sequence 5'-dGAb.sup.6 GCT-3'
wherein b.sup.6 G is a substituted or unsubstituted O.sup.6
-benzylguanine.
41. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 40 and a pharmaceutically
acceptable carrier.
42. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 -position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide of claim 40.
43. A single stranded oligodeoxyribonucleotide that inactivates
human alkyltransferase consisting of 5 to 11 bases at least one of
which is a substituted or unsubstituted O.sup.6 -benzylguanine.
44. A composition comprising the single-stranded
oligodeoxyribonucleotide of claim 43 and a pharmaceutically
acceptable carrier.
45. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 -position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal, which method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyripbonucleotide of claim 43.
46. A method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 -position of guanine
residues in DNA, in the chemotherapeutic contacting of cancer cells
in vitro, which method comprises co-administering to the cancer
cells an antineoplastic alkylating agent and a single-stranded
oligodeoxyribonucleotide of claim 1.
47. A single-stranded oligodeoxyribonucleotide of not less than 5
and not more than 11 substituted or unsubstituted bases in length
comprising at least one substituted or unsubstituted O.sup.6
-benzylguanine base, and which inactivates human
alkyltransferase.
48. The single-stranded oligodeoxyribonucleotide of claim 47,
wherein at least one of the 5 to 11 bases other than the
substituted or unsubstituted O.sup.6 -benzylguanine base is
substituted.
49. The single-stranded oligodeoxyribonucleotide of claim 47,
comprising at least one modified internucleoside phosphate
linkage.
50. The single-stranded oligodeoxyribonucleotide of claim 49,
comprising two or more modified internucleoside phosphate
linkages.
51. The single-stranded oligodeoxyribonucleotide of claim 49,
wherein said modified internucleoside phosphate linkage is a
modified terminal internucleoside phosphate linkage.
52. The single-stranded oligodeoxyribonucleotide of claim 51,
wherein said modified terminal internucleoside phosphate linkage is
a methylphosphonate or a phosphorothioate.
53. A modified single-stranded oligodeoxyribonucleotide of not less
than 5 and not more than 11 substituted or unsubstituted bases in
length comprising at least one substituted or unsubstituted O.sup.6
-benzylguanine base, and which inactivates human
alkyltransferase.
54. The single-stranded oligodeoxyribonucleotide of claim 1,
including a plurality of substituted or unsubstituted O.sup.6
-benzylguanine bases.
55. The single-stranded oligodeoxyribonucleotide of claim 54,
including at least three substituted or unsubstituted O.sup.6
-benzylguanine bases.
56. The single-stranded oligodeoxyribonucleotide of claim 54,
including at least four substituted or unsubstituted O.sup.6
-benzylguanine bases.
57. The single-stranded oligodeoxyribonucleotide of claim 54,
including at least five substituted or unsubstituted O.sup.6
-benzylguanine bases.
58. The single-stranded oligodeoxyribonucleotide of claim 2,
including a plurality of substituted or unsubstituted O.sup.6
-benzylguanine bases.
59. The single-stranded oligodeoxyribonucleotide of claim 58,
including at least three substituted or unsubstituted O.sup.6
-benzylguanine bases.
60. The single-stranded oligodeoxyribonucleotide of claim 58,
including at least four substituted or unsubstituted O.sup.6
-benzylguanine bases.
61. The single-stranded oligodeoxyribonucleotide of claim 58,
including at least five substituted or unsubstituted O.sup.6
-benzylguanine bases.
Description
TECHNICAL FIELD OF THE INVENTION
This invention relates to oligodeoxyribonucleotides comprising
O.sup.6 -benzylguanine and related compositions. This invention
also relates to the use of such oligodeoxyribonucleotides and
related compositions to enhance the effect of an antineoplastic
alkylating agent in the chemotherapeutic treatment of cancer in a
mammal.
BACKGROUND OF THE INVENTION
O.sup.6 -alkylguanine-DNA alkyltransferase (AGT) is a DNA repair
protein. AGT removes alkyl and aralkyl groups that become attached
at the O.sup.6 position of guanine in DNA or alkyl groups at the
O.sup.4 position of thymine in DNA following exposure to mutagenic
and/or carcinogenic alkylating agents. It does so by bringing about
a stoichiometric transfer of the group attached to the O.sup.6
position of a guanine residue in DNA, for example, to a cysteine
residue within the AGT protein (Pegg, Cancer Research 50: 6119-6129
(1990)). Accordingly, AGT is beneficial to a normal cell because it
removes the adducts that are formed in DNA by toxic, mutagenic and
carcinogenic agents, thereby restoring the DNA to its original
state and helping to prevent DNA mutations that can lead to
initiation of tumor formation. Unfortunately, AGT is also
beneficial to a cancerous cell because it also removes those
adducts that are formed at the O.sup.6 position of guanine in DNA
by antineoplastic alkylating agents, such as monofunctional
methylating agents, e.g., procarbazine, dacarbazine and
temozolomide, and chloroethylating agents, i.e., CENUs, such as
BCNU, ACNU, CCNU, MeCCNU, fotemustine and clomesone (Pegg et al.,
Prog. Nucleic Acid Research Molec. Biol. 51: 167-223 (1995)). The
resulting alkylated AGT molecule is consequently inactivated and is
unable to carry out subsequent dealkylation reactions. The presence
of more AGT in a cell increases its capacity to repair DNA by this
mechanism compared to a cell that has less AGT.
The reduction in the efficacy of cancer chemotherapeutic drugs due
to AGT, which acts without requiring the presence of additional
enzymes or cofactors, and the existence of a high correlation
between AGT activity and reduction in sensitivity of tumor cells to
nitrosoureas have led to AGT becoming a prime target for
modulation. Modulation has been attempted by two different routes.
One route is indirect and involves the use of methylating agents
that introduce O.sup.6 -methylguanine lesions into DNA for
subsequent repair by AGT, thereby depleting levels of AGT. The
other route is direct and involves the use of an inactivator of
AGT, such as an O.sup.6 -aralkylguanine (see, for example, Moschel
et al., U.S. Pat. Nos. 5,091,430, 5,352,669 and 5,358,952).
The first O.sup.6 -alkylguanine developed as a potential
inactivator of AGT was O.sup.6 -methylguanine. Although initial
results obtained in cell culture appeared promising, O.sup.6
-methylguanine was only able to reduce AGT activity by 85% and was
not able to enhance the therapeutic index of BCNU in the treatment
of mice carrying human tumor xenografts (Pegg et al. (1995),
supra). In addition, the use of O.sup.6 -methylguanine was plagued
with problems, such as poor solubility, poor affinity for AGT, poor
uptake into cells, and lack of selectivity, which necessitated high
dosages of O.sup.6 -methylguanine to be administered for long
periods of time (Pegg et al. (1995), supra).
The testing of O.sup.6 -methylguanine led to the development of
O.sup.6 -benzylguanine as a potential inactivator of AGT (Moschel
et al., J. Med. Chem. 35(23): 4486-4491 (1992); Pegg et al.,
Biochem. 32(45): 11998-12006 (1993); Pegg et al., Proc. Amer.
Assoc. Cancer Research 34: 565 (1993); and Gerson et al., Proc.
Amer. Assoc. Cancer Research 35: 699 (1994)). O.sup.6
-benzylguanine has been shown to inactivate AGT in Mer.sup.30
cells, thereby rendering them more sensitive to the cytotoxic
effects of alkylating agents (Pegg et al. (1995), supra).
Furthermore, the correlation between the degree of increased
sensitivity to alkylating agents and the level of inhibition of AGT
activity by O.sup.6 -benzylguanine is strong (Pegg et al. (1995),
supra). O.sup.6 -benzylguanine also has been shown to increase the
sensitivity of oxic and hypoxic brain tumor cells to BCNU (Pegg et
al. (1995), supra). Increased sensitivity to MeCCNU or BCNU due to
the prior administration of O.sup.6 -benzylguanine also was
demonstrated in nude mice carrying SF767 tumor xenografts (Dolan et
al., Cancer Comm. 2(11): 371-377 (1990)), mice carrying a D341MED
or a D456MG brain tumor xenograft or a TE-671 human rhabdosarcoma
xenograft (Pegg et al. (1995), supra; Friedman et al., J. Natl.
Cancer Inst. 84(24): 1926-1931 (1992); and Felker et al., Cancer
Chemother. Pharmacol. 32: 471-476 (1993)). A significant increase
in median survival in animals treated with O.sup.6 -benzylguanine
prior to BCNU compared to BCNU alone was demonstrated in the
intracranial D341 MED medulloblastoma model (Pegg et al. (1995),
supra; and Friedman et al. (1992), supra). Similar observations
have been made with respect to colon tumor xenografts having high
AGT activity (Mitchell et al., Cancer Research 52: 1171-1175
(1992); and Dolan et al., Biochem. Pharmacol. 46(2): 285-290
(1993)) and the Dunning rat prostate tumor model (Pegg et al.
(1995), supra; and Dolan et al., Cancer Chemother. Pharmacol. 32:
221-225 (1993)). Exogenously added DNA, such as single-stranded and
double-stranded oligodeoxyribonucleotides ranging in length from 4
to 16 bases (or base pairs), in particular 12-base (or 12-base
pair) oligodeoxyribonucleotides, have been shown to stimulate the
production of guanine by recombinant human AGT from O.sup.6
-benzylguanine, but not 9-substituted O.sup.6 -benzylguanines
(Goodtzova et al., Biochem. 33(28): 8385-8390 (1994)).
p-Chlorobenzyl and p-methylbenzyl analogues of O.sup.6
-benzylguanine also have been shown to inactivate AGT rapidly and
irreversibly (Dolan et al., PNAS USA 87: 5368-5372 (1990); and
Dolan et al., Cancer Research 51: 3367-3372 (1991)). Such analogues
have been shown to be as good as O.sup.6 -benzylguanine in
enhancing the cytotoxicity of chloroethylating agents toward SF767
glioma cells and HT29 colon tumor cells (Dolan et al. (1990),
supra; and Dolan et al. (1991), supra). Based on such results,
O.sup.6 -benzylguanine was suggested to be potentially useful in
the treatment of mer+ tumors as an adjuvant to an alkylating agent
that produces a toxic lesion at the O.sup.6 position of guanine
residues in DNA (Dolan et al. (1991), supra).
O.sup.6 -benzylguanine, in combination with BCNU, is now in
clinical trials. Although O.sup.6 -benzylguanine is clearly the
most promising compound for inactivating AGT at this time, it is
not an ideal drug. It has only limited solubility in water and is
characterized by rapid clearance from blood plasma due to metabolic
conversion to other compounds (Dolan et al., Cancer Research 54:
5123-5130 (1994)).
Furthermore, in vitro data suggest that O.sup.6 -benzylguanine may
not be able to inactivate mutant forms of AGT, which could result
from mutations induced by chemotherapeutic drugs, such as
chloroethylating or methylating agents, in vivo. Given that the E.
coli Ada-C protein and Ogt alkyltransferase and the yeast AGT are
insensitive to O.sup.6 -benzylguanine (Pegg et al., Biochem. 32:
11998-12006 (1993); and Elder et al., Biochem. J. 298: 231-235
(1994)), site-directed mutagenesis (Crone et al., Cancer Research
53: 4750-4753 (1993); Crone et al., Cancer Research 54: 6221-6227
(1994); and Edara et al., Cancer Research 56: 5571-5575 (1996)) has
been used to create mutant AGTs, which differ from the wild-type
AGT by one or more amino acid changes. Several mutant AGTs have
been found to be much less sensitive than wild-type AGT to
inactivation by O.sup.6 -benzylguanine (Crone et al. (1993), supra;
Crone et al. (1994), supra; and Edara et al. (1996), supra).
In an effort to address its limited solubility in water, O.sup.6
-benzylguanine has been formulated in a polyethylene
glycol-400-based vehicle (Pegg et al. (1995), supra). The
formulation has been shown to be effective in sensitizing D456MG
glioblastoma xenografts in nude mice to BCNU at lower doses than
earlier cremophor-EL-based formulations (Pegg et al. (1995),
supra). significantly more effective than O.sup.6 -benzylguanine at
inactivating AGT in human HT29 colon tumor cell extracts and intact
HT29 colon tumor cells (Chae et al., J Med Chem. 38: 359-365
(1995)). Consequently, it has been suggested that these new
compounds may be superior to O.sup.6 -benzylguanines as
chemotherapeutic adjuvants for enhancing the effectiveness of
antitumor drugs that modify the O.sup.6 -position of guanine
residues in DNA (Chae et al. (1995), supra). However, some of the
pyrimidines appear to be metabolized and rapidly excreted (Roy et
al., Drug Metab. Disposition, 24: 1205-1211 (1996)).
Sixteen-base oligonucleotides comprising one or two O.sup.6
-methyl-, O.sup.6 -ethyl- or O.sup.6 -benzyl-2'-deoxyguanosine
residue(s) have been generated. These have the sequences of the rat
H-ras gene extending from codon 9 through the first base of codon
14. These oligonucleotides were used to establish whether the type
of O.sup.6 -substituted 2'-deoxyguanosine residue or its position
leads to any significant differential disruption of duplex
stability or conformation that might ultimately contribute to a
rationale for the apparent selective mutability of the second
guanine residue of codon 12 of H-ras in rat mammary carcinomas upon
activation following a single dose of NMU (Pauly et al., Chem.
Research Toxicol. 1(6): 391-398 (1988)). Related sixteen-base
oligonucleotides also have been incorporated into a cassette
plasmid for use in E. coli to monitor the mutagenicity of
carcinogen-modified bases in a simple sectored colony assay (Pauly
et al., Biochemistry 30: 11700-11706 (1991)) and to compare their
repair by mammalian and bacterial AGTs (Elder et al., Biochem. J.
298: 231-235 (1994)). An example of such a sixteen-base
oligonucleotide was found to deplete AGT activity rapidly and has
been described as a possibly good substrate for AGT (Dolan et al.
(1990), supra).
In view of the above, there remains a need for an inhibitor of AGT,
which (i) is more water-soluble than O.sup.6 -benzylguanine, (ii)
is effective at a much lower concentration than O.sup.6
-benzylguanine, (iii) is capable of inactivating mutant forms of
AGT, which are resistant to inactivation by O.sup.6 -benzylguanine,
and (iv) is still more active than O.sup.6 -methylguanine and
analogues thereof. Accordingly, it is an object of the present
invention to provide such an inactivator. It is another object of
the present invention to provide a composition comprising such an
inactivator. It is yet another object of the present invention to
provide a method of using such inactivators and compositions. These
and other objects will become apparent from the detailed
description set forth below.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a single-stranded
oligodeoxyribonucleotide, which (i) comprises from about 5 to 11
bases, at least one of which is a substituted or an unsubstituted
O.sup.6 -benzylguanine, and (ii) inactivates human AGT. The present
invention also provides a single-stranded oligodeoxyribonucleotide,
which can inactivate a mutant human AGT, which either is not
inactivated by O.sup.6 -benzylguanine or is less inactivated by
O.sup.6 -benzylguanine than by said single-stranded
oligodeoxyribonucleotide. One or more phosphates of the
single-stranded oligodeoxyribonucleotide can be modified, e.g., by
replacement with a methylphosphonate or a phosphorothioate. The
present invention also provides a composition comprising such an
oligodeoxyribonucleotide. In addition, the present invention
provides a method of enhancing the effect of an antineoplastic
alkylating agent, which alkylates the O.sup.6 position of guanine
residues in DNA, in the chemotherapeutic treatment of cancer in a
mammal. The method comprises the co-administration to the mammal of
a cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
present inventive oligodeoxyribonucleotide or a composition
thereof.
BRIEF DESCRIPTION OF THE DRAWING
FIGS. 1A-C are graphs of percent remaining alkyltransferase
activity (% AGT activity remaining) versus concentration of a
single-stranded oligodeoxyribonucleotide (3-11 nts in length,
designated 3-mer, 5-mer, 7-mer, 9-mer and 11-mer) comprising
O.sup.6 -benzylguanine (nM Inhibitor) for wild-type human
alkyltransferase (AGT, FIG. 1A) and mutant human alkyltransferases
(G156A, FIG. 1B, and P140A, FIG. 1C).
DETAILED DESCRIPTION OF THE INVENTION
The present invention is predicated on the discovery that a
single-stranded oligodeoxyribonucleotide, which comprises from
about 5 to 11 bases, at least one of which is a substituted or an
unsubstituted O.sup.6 -benzylguanine, is a more effective
inactivator of human AGT than the free base O.sup.6 -benzylguanine.
Not only is the single-stranded
oligodeoxyribonucleotide more effective in inactivating human AGT,
it does not suffer from the major disadvantage attendant the free
base O.sup.6 -benzylguanine, namely its limited solubility in
water. Another characteristic that distinguishes the
single-stranded oligodeoxyribonucleotide from the free base O.sup.6
-benzylguanine is its ability to inactivate mutant human AGTs,
which could be produced by various chemotherapeutic drugs, such as
chloroethylating or methylating agents, in vivo and which either
are not inactivated by O.sup.6 -benzylguanine or are less
inactivated by O.sup.6 -benzylguanine than by the single-stranded
oligodeoxyribonucleotide. In addition, the present inventive
single-stranded oligodeoxyribonucleotides have been designed so
that they are no longer than necessary to fit the active site of
the human AGT, thereby rendering them easier to synthesize and
purify than comparatively longer single-stranded
oligodeoxyribonucleotides and double-stranded
oligodeoxyribonucleotides.
In view of the above, the present invention provides a
single-stranded oligodeoxyribonucleotide, which (i) comprises from
about 5 to 11 bases, at least one of which is a substituted or an
unsubstituted O.sup.6 -benzylguanine, and (ii) inactivates human
AGT. Preferably, the single-stranded oligodeoxyribonucleotide
comprises from about 7 to 11 bases, more preferably from about 9 to
11 bases. Examples of such oligodeoxyribonucleotides are set forth
in Table I.
Although it is only necessary that a single base in the
single-stranded oligodeoxyribonucleotide be a substituted or an
unsubstituted O.sup.6 -benzylguanine, as many as two, three, four
or even every base in the single-stranded oligodeoxyribonucleotide
can be a substituted or an unsubstituted O.sup.6 -benzylguanine. If
there is more than one substituted or unsubstituted O.sup.6
-benzylguanine present in the single-stranded
oligodeoxyribonucleotide, they can be the same or different. A
preferred single-stranded oligodeoxyribonucleotide is one in which
the middle base is a substituted or an unsubstituted O.sup.6
-benzylguanine.
Although the at least one O.sup.6 -benzylguanine is preferably
unsubstituted, the O.sup.6 -benzylguanine can be substituted. The
manner in which the O.sup.6 -benzylguanine is substituted and the
extent to which the O.sup.6 -benzylguanine is substituted is not
narrowly critical to the practice of the present invention. All
that matters is that the resulting single-stranded
oligodeoxyribonucleotide inhibits human AGT.
Desirably, the single-stranded oligodeoxyribonucleotide inactivates
human AGT more effectively than the free base O.sup.6
-benzylguanine. Preferably, the single-stranded
oligodeoxyribonucleotide inactivates a mutant human AGT, which
could be produced by various chemotherapeutic drugs, such as
chloroethylating or methylating agents, in vivo and which either is
not inactivated by O.sup.6 -benzylguanine or is less inactivated by
O.sup.6 -benzylguanine than by the single-stranded
oligodeoxyribonucleotide.
If the O.sup.6 -benzylguanine is substituted, preferably it is
substituted with from one to five substituents, which can be the
same or different and are hydro, halo, haloalkyl, hydroxy,
hydroxyamino, hydrazino, an alkyl, an aryl, nitro, a polycyclic
aromatic alkyl, a cycloalkyl, an alkenyl, an alkynyl, a
hydroxyalkyl, an alkoxy, an alkoxylalkyl, an aryloxy, an acyloxy,
an acyloxyalkyl, a monoalkylamino, a dialkylamino, an acylamino, an
ureido, a thioureido, a carboxy, a carboxyalkyl, a cyano, a
cyanoalkyl, C-formyl, C-acyl, a dialkoxyalkyl, or SO.sub.n R.sub.1,
wherein n is an integer from zero to three and R.sub.1 is hydro, a
C.sub.1 -C.sub.6 alkyl, or a C.sub.1 -C.sub.4 alkyl-substituted or
an unsubstituted aryl. The one to five substituents are
independently substituted or unsubstituted. More preferably, the
haloalkyl is a C.sub.2 -C.sub.6 straight-chain or a C.sub.3
-C.sub.6 branched-chain alkyl, which is substituted with from one
to three halo groups, the alkyl is a C.sub.2 -C.sub.6
straight-chain or a C.sub.3 -C.sub.6 branched-chain alkyl, the aryl
is substituted with a C.sub.1 -C.sub.8 straight-chain or a C.sub.3
-C.sub.8 branched-chain alkyl, the polycyclic aromatic alkyl
comprises from two to four aromatic rings and a C.sub.1-C.sub.6
straight-chain or a C.sub.3 -C.sub.6 branched-chain alkyl, the
cycloalkyl is a C.sub.3 -C.sub.8 cycloalkyl, the alkenyl is a
C.sub.2 -C.sub.6 straight-chain or a C.sub.4 -C.sub.6
branched-chain alkenyl, the alkynyl is a C.sub.2 -C.sub.6
straight-chain or a C.sub.4 -C.sub.6 branched-chain alkynyl, the
hydroxyalkyl is a C.sub.1-C.sub.6 straight-chain or a C.sub.3
-C.sub.6 branched-chain hydroxyalkyl, the alkoxy is a C.sub.1
-C.sub.8 straight-chain or a C.sub.3 -C.sub.8 branched-chain
alkoxy, the alkoxyalkyl is a C.sub.2 -C.sub.8 alkoxyalkyl, the
acyloxyalkyl comprises a C.sub.1 -C.sub.6 straight-chain or a
C.sub.3 -C.sub.6 branched-chain alkyl, the monoalkylamino and the
dialkylamino comprise a C.sub.1 -C.sub.6 straight-chain or a
C.sub.3 -C.sub.6 branched-chain alkyl, the carboxyalkyl comprises a
C.sub.1 -C.sub.6 straight-chain or a C.sub.3 -C.sub.6
branched-chain alkyl, the cyanoalkyl comprises a C.sub.1 -C.sub.6
straight-chain or a C.sub.3 -C.sub.6 branched-chain alkyl, and the
dialkoxyalkyl comprises a C.sub.1 -C.sub.6 straight-chain or a
C.sub.3 -C.sub.6 branched-chain alkoxy, which can be the same or
different, and a C.sub.1 -C.sub.6 straight-chain or a C.sub.3
-C.sub.6 branched-cha
O.sup.6 -benzylguanine can be prepared in accordance with the
method set forth in Bowles et al., J. Med. Chem. 6: 471-480 (1963),
or Frihart et al., J. Am. Chem. Soc. 95: 7144-7175 (1973).
Substituted O.sup.6 -benzylguanines can be synthesized by reacting
2-amino-6-chloropurine with the alkoxide of a benzyl alcohol
comprising a desired ortho, meta orpara substituent. Procedures
that can be used to prepare substituted O.sup.6 -benzylguanine are
set forth in Dolan et al. (1990), supra. Treatment of O.sup.6
-benzylguanine in its anionic form with alkylating agents, such as
ethyl bromoacetate, 2-bromoacetamide, 1,2-epoxybutane,
bromoacetonitrile or
1,3,4,6-tetra-O-acetyl-2-deoxy-2-(chloroacetamido)-.beta.-D-glucose,
are described in Moschel et al. (1992), supra, and Fondy et al., J.
Med Chem. 21: 1222-1225 (1978). .alpha.-amino acid adducts of
O.sup.6 -benzylguanine can be prepared by nucleophilic displacement
by O.sup.6 -benzylguanine or its anion on selected reagents, such
as the protected .beta.-lactone of L-serine (Pansare et al., Org.
Syn. 70: 1-9 (1991)) or (S)-3-amino-2-oxetanone (Pansare et al.,
Org. Syn. 70: 10-17 (1991)). Specific examples of synthesis of
substituted O.sup.6 -benzylguanine are set forth in Moschel et al.,
U.S. Pat. No. 5,691,307.
Single-stranded oligodeoxyribonucleotides comprising at least one
O.sup.6 -benzylguanine can be synthesized in accordance with
methods known to those of ordinary skill in the art. For example,
automated DNA synthetic procedures can be used to introduce a
suitably protected phosphoramidite of O.sup.6
-benzyl-2'-deoxyguanosine into a DNA sequence at any location
(Pauly et al. (1988), supra; and Pauly et al. (1991), supra). If
desired, O.sup.6 -benzylguanine can be attached through a linker to
a hydroxyl group at a terminal carbohydrate residue of an
oligodeoxyribonucleotide, for example, by reacting
2-amino-6-benzyloxy-9-carboethoxymethylpurine with the hydroxyl
group of the terminal carbohydrate residue of the
oligodeoxyribonucleotide.
The above-described single-stranded oligodeoxyribonucleotide can be
modified, for example, to increase its resistance to nuclease
digestion in vivo. In this regard, at least one, although it can be
more, phosphate is modified, preferably by replacement with a
methylphosphonate or a phosphorothioate. More preferably, at least
one of either of the terminal phosphates is replaced by a
methylphosphonate or a phosphorothioate. Even more preferably, both
of the terminal phosphates are independently replaced by a
methylphosphonate or a phosphorothioate, i.e., the replacements can
be the same or different. Such modifications are within the
ordinary skill in the art (see, for example, Marcus-Sekura et al.,
Nucleic Acids Research 15: 5749-5763 (1987) and references cited
therein). Care should be exercised to ensure that not so many
phosphates in any given oligodeoxyribonucleotide are modified so as
to affect adversely the ability of the oligodeoxyribonucleotide to
inactivate human AGT.
Desirably, the single-stranded oligodeoxyribonucleotide also
inactivates a mutant human alkyltransferase. Preferably, the mutant
human alkyltransferase is one that either is not inactivated by
O.sup.6 -benzylguanine or is less inactivated by O.sup.6
-benzylguanine than by the single-stranded
oligodeoxyribonucleotide.
Whether or not a given single-stranded oligodeoxyribonucleotide
inactivates a wild-type or mutant human alkyltransferase can be
determined by measuring alkyltransferase depletion. For example, a
stock solution (100 mM) of a given single-stranded
oligodeoxyribonucleotide in an aqueous or mixed aqueous/organic
solvent can be prepared. Solutions of the human wild-type AGT,
mutant human AGT, or the AGT from HT29 cells and cell extracts
(Domoradzki et al., Carcinogenesis 5: 1641-1647 (1984)) can be
incubated with varying concentrations (between 0 and 400 .mu.M, for
example) of the oligodeoxyribonucleotide for 30 min in a buffer
containing 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, and 5 mM
dithiothreitol, and alkyltransferase depletion can be measured.
Alternatively, cells can be plated at a density of 5.times.10.sup.6
cells/T75 flask and allowed to grow for three days, at which time
the medium can be replaced with medium containing a given
concentration of an oligodeoxyribonucleotide. After four hours,
cells can be harvested and frozen at -80.degree. C. for analysis of
alkyltransferase depletion later. Alkyltransferase depletion is
determined by measuring loss of O.sup.6 -[.sup.3 H]methylguanine,
for example, from a [.sup.3 H]methylated DNA substrate, for
example, which can be prepared by reacting [.sup.3
H]methylnitrosourea (21.5 Ci/mmol) with calf thymus DNA as
described previously (Domoradzki et al. (1984), supra; and Dolan et
al. (1990), supra).
Any oligodeoxyribonucleotide in accordance with the present
invention that effectively depletes tumor cells of alkyltransferase
activity as measured, for example, in the above-described assay, is
expected to enhance the effect of an antineoplastic alkylating
agent, which alkylates the O.sup.6 the position of guanine residues
in DNA, in the chemotherapeutic treatment of cancer in a mammal.
This has been shown to be true with the weak alkyltransferase
depleter O.sup.6 -benzylguaninemethylguanine (Dolan et al., Cancer
Research 46: 4500-4504 (1986)) and the more potent alkyltransferase
depleters O.sup.6 -benzylguanine and O.sup.6 -(p-chlorobenzyl)-and
O.sup.6 -(p-methylbenzyl)-guanine (Dolan et al. (1990), supra;
Dolan et al. (1991), supra; Dolan et al. (1993), supra; Mitchell et
al., Cancer Research 52: 1171-1175 (1992); and Moschel et al., U.S.
Pat. No. 5,691,307).
In addition to the above-described oligodeoxyribonucleotides, the
present invention also provides a composition comprising a
single-stranded oligodeoxyribonucleotide and a pharmaceutically
acceptable carrier. Appropriate pharmaceutically acceptable
carriers, vehicles, adjuvants, excipients and diluents are known in
the art. The above-described oligodeoxyribonucleotides or
pharmaceutically acceptable salts thereof can be formulated into
solid, semi-solid, liquid or gaseous formulations. Examples of such
formulations include tablets, capsules, powders, granules,
ointments, solutions, suppositories, injections, inhalants and
aerosols. The choice of formulation will be determined, in part, by
the particular route of administration chosen. The following
formulations are merely exemplary and are, in no way, limiting.
Compositions for oral administration (also, buccal or sublingual)
can comprise additives, such as lactose, mannitol, corn starch or
potato starch, binders, such as crystalline cellulose, cellulose
derivatives, acacia, corn starch or gelatin, with sodium
carboxymethylcellulose, lubricants, such as talc or magnesium
stearate, and, if desired, diluents, buffering agents, moistening
agents, preservatives and flavoring agents. Such compositions can
be in the form of tablets, powders, granules or capsules, for
example. Unit dosage forms for oral administration, such as syrups,
elixirs and suspensions, wherein each dosage unit, e.g.,
teaspoonful or tablespoonful, contains a predetermined amount of a
present inventive oligodeoxyribonucleotide, can be combined with
sterile water for injection (USP) or normal saline.
Compositions for administration in the form of suppositories can
comprise a base. Suitable bases include emulsifying bases and
water-soluble bases. Vehicles, such as cocoa butter, carbowaxes and
polyethylene glycols, which are solid at room temperature and melt
at body temperature, also can be used.
Compositions for transdermal administration comprise an appropriate
vehicle or salt. Adsorption can be aided by the use of an electric
current or field.
Compositions for administration by injection can be prepared by
dissolving, suspending or emulsifying a present inventive
oligodeoxyribonucleotide in an aqueous or nonaqueous solvent, such
as vegetable oil, synthetic aliphatic acid glycerides, esters of
higher aliphatic acids or propylene glycol. Solubilizers, isotonic
agents, suspending agents, emulsifying agents, stabilizers and
preservatives can be added, if desired.
Compositions for aerosolized administration can be prepared in the
form of a liquid or minute powder. An aerosol container can be
filled with gaseous or liquid spraying agents and, if desired,
conventional adjuvants, such as humidifying agents. Suitable
propellants include dichlorodifluoromethane, propane, nitrogen and
the like. If desired, the composition can be formulated for
non-pressurized preparations, such as a nebulizer or an
atomizer.
In view of the above, the present invention also provides a method
of enhancing the effect of an antineoplastic alkylating agent,
which alkylates the O.sup.6 position of guanine residues in DNA, in
the chemotherapeutic treatment of cancer in a mammal, particularly
a human. The method comprises co-administering to the mammal a
cancer-treatment effective amount of an antineoplastic alkylating
agent and a chemotherapeutic treatment-enhancing amount of a
single-stranded oligodeoxyribonucleotide in accordance with the
present invention.
By "enhancing the effect of an antineoplastic alkylating agent" is
meant that the antineoplastic alkylating agent has a greater effect
in the presence of a present inventive oligodeoxyribonucleotide
than in the absence of a present inventive
oligodeoxyribonucleotide. When an alkyltransferase acts on the
oligodeoxyribonucleotide, it is inactivated and, therefore, is not
able to act on the DNA in a cancerous cell that has been alkylated
by the antineoplastic alkylating agent. Given that the
alkyltransferase is not able to act on the alkylated DNA in a
cancerous cell, the DNA in the cancerous cell is not repaired,
thereby leading to death of the cancerous cell.
By "coadministering" is meant administering the antineoplastic
alkylating agent and the oligodeoxyribonucleotide sufficiently
close in time such that the oligodeoxyribonucleotide can enhance
the effect of the antineoplastic alkylating agent. In this regard,
the oligodeoxyribonucleotide can be administered first and the
antineoplastic alkylating agent can be administered second or vice
versa. Alternatively, the oligodeoxyribonucleotide and the
antineoplastic alkylating agent can be administered simultaneously.
In addition, a combination of oligodeoxyribonucleotides can be
administered, and one or more oligodeoxyribonucleotides can be
administered in combination with another agent useful in the
treatment of cancer.
By "cancer-treatment effective amount of an antineoplastic
alkylating agent" is meant that the antineoplastic alkylating agent
is administered in a dose sufficient to treat the cancer. Such
doses are known in the art (see, for example, the Physicians' Desk
Reference). For example, 1,3-bis(2-chloroethyl)-1-nitrosourea
(carmustine or BCNU, Bristol-Myers, Evansville, Ind.) can be
administered intravenously at a dosage of from about 150 to 200
mg/m.sup.2 every six weeks. Another alkylating agent, namely
1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (lomustine or CCNU,
Bristol-Myers), can be administered orally at a dosage of about 130
mg/m.sup.2 every six weeks. By "chemotherapeutic
treatment-enhancing amount of a single-stranded
oligodeoxyribonucleotide" is meant that the
oligodeoxyribonucleotide is administered in a dose sufficient to
enhance the effect of the antineoplastic alkylating agent. A
suitable dosage is that which will result in a concentration of
oligodeoxyribonucleotide in
the cancerous cells to be treated sufficient to deplete
alkyltransferase activity, e.g., from about 10 nM to 200 nM
intracellularly, which may require an extracellular concentration
of from about 10 .mu.M to 50 .mu.M. The dose can be adjusted as
necessary to enhance the effect of the antineoplastic alkylating
agent.
The present inventive oligodeoxyribonucleotides are useful in
enhancing the effect of any antineoplastic alkylating agent,
provided that the agent is one that alkylates the O.sup.6 position
of guanine residues in DNA. Examples of antineoplastic alkylating
agents include chloroethylating agents. The most frequently used
chloroethylating agents include
1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU, lomustine),
1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU, carmustine),
1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea (MeCCNU,
semustine), and
1-(2-chloroethyl)-3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-nitrosourea
(ACNU). Such agents have been used clinically against tumors of the
central nervous system, multiple myeloma, melanoma, lymphoma,
gastrointestinal tumors, and other solid tumors (Colvin and
Chabner. Alkylating Agents. In: Cancer Chemotherapy: Principles and
Practice. Edited by B. A. Chabner and J. M. Collins, Lippincott,
Philadelphia, Pa. pp. 276-313 (1990); and McCormick et al., Eur. J.
Cancer 26: 207-221 (1990)). Chloroethylating agents, which have
fewer side effects and are currently under development include
1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea (HECNU),
2-chloroethylmethylsulfonylmethanesulfonate (Clomesone), and
1-[N-(2-chloroethyl)-N-nitrosoureido]ethylphosphonic acid diethyl
ester (Fotemustine) (Colvin and Chabner (1990), supra; and
McCormick et al. (1990), supra). Methylating agents include
Streptozotocin
(2-deoxy-2-(3-methyl-3-nitrosoureido)-D-glucopyranose),
Procarbazine
(N-(1-methylethyl)-4-[(2-methylhydrazino)methyl]benzamide),
Dacarbazine or DTIC
(5-(3,3-dimethyl-1-triazenyl)-1H-imidazole-4-carboxamide), and
Temozolomide
(8-carbamoyl-3-methylimidazo[5.1-d]-1,2,3,5-tetrazin-4-(3H)-one).
Temozolomide is active against malignant melanomas, brain tumors
and mycosis fungoides. Streptozotocin is effective against
pancreatic tumors. Procarbazine is used to treat Hodgkin's disease
and brain tumors. DTIC is used to treat melanoma and lymphomas
(Colvin and Chabner (1990), supra; and Longo, Semin. Concol. 17:
716-735 (1990)).
The antineoplastic alkylating agent can be administered by any
route. Conventional means of administration are described in
Wasserman et al. (Cancer 36: 1258-1268 (1975)) and in the
Physicians' Desk Reference (44.sup.th ed., Edward R. Barnhart,
publisher, 1990.).
The present inventive method can be used to treat any cancer
susceptible to treatment by an antineoplastic alkylating agent.
Examples of such cancers include prostate cancer, brain cancer,
lymphoma, leukemia, breast cancer, ovarian cancer, lung cancer,
Wilms' tumor, rhabdomyosarcoma, multiple myeloma, stomach cancer,
soft-tissue sarcoma, Hodgkin's disease, and non-Hodgkin's
lymphoma.
The following example further illustrates the present invention.
The example, of course, should not be construed as in any way
limiting the scope of the present invention.
O.sup.6 -benzylguanine was synthesized as previously described
(Dolan et al. (1990), supra). Single-stranded
oligodeoxyribonucleotides also were synthesized in accordance with
methods described by Pauly et al. (1991), supra. O.sup.6
-benzylguanine was purified by crystallization from water and the
oligodeoxyribonucleotides were purified by HPLC (Pauly et al.
(1988), supra). The composition of the oligodeoxyribonucleotides
was confirmed by enzymatic digestion to 2'-deoxyribonucleosides
(Pauly et al. (1988), supra).
EXAMPLE
This example demonstrates that wild-type and mutant human
alkyltransferases are more sensitive to inactivation by a
single-stranded oligodeoxyribonucleotide comprising O.sup.6
-benzylguanine than by O.sup.6 -benzylguanine, itself.
Wild-type human AGT and mutants thereof, namely P140A and G156A,
were prepared using the pIN vector expression system. pIN-AGT,
pIN-P140A (Pro-140 to Ala) and pIN-G156A (Gly-156 to Ala) (Crone et
al. (1993), supra; Crone et al. (1994), supra; and Pegg et al.
(1993), supra).
The wild-type and mutant AGT proteins expressed from the pIN
vectors were purified to homogeneity by ammonium sulfate
precipitation, Mono-S chromatography and gel filtration as
previously described (Pegg et al. (1993), supra; and Kanugula et
al. (1995), supra). The purified protein was then incubated with
O.sup.6 -benzylguanine, O.sup.6 -benzyl-2'-deoxyguanosine, or a
single-stranded oligodeoxyribonucleotide ranging in length from
three to eleven bases and comprising O.sup.6 -benzylguanine in 0.1
ml of 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA and 5.0 mM dithiothreitol
for 30 min at 37.degree. C. Afterwards, residual AGT activity was
determined by a 30 min incubation with a [.sup.3 H]-methylated DNA
substrate (1.0 ml volume), which had been methylated by reaction
with N-[.sup.3 H]-methyl-N-nitrosourea as previously described
(Dolan et al. (1993), supra; and Dolan et al. (1991), supra).
The results were expressed as the percentage of the AGT activity
remaining and then used to calculate the ED.sub.50 value (the
concentration needed to reduce AGT activity by 50%) for the
inactivator as shown in Table I.
TABLE I ______________________________________ ED.sub.50 for
inactivation of AGT (nM) Oligodeoxyribonucleotide AGT G156A P140A
______________________________________ O.sup.6 -benzylguanine.sup.a
200 60,000 5,000 O.sup.6 -benzyl-2'-deoxyguanosine.sup.b 2000
>100,000 >20,000 5'-d(Ab.sup.6 GC)-3' 90 4600 770
5'-d(GAb.sup.6 GCT)-3' 13 60 50 5'-d(TGAb.sup.6 GCTG)-3' 7 50 60
5'-d(GTGAb.sup.6 GCTGT)-3' 8 90 60 5'-d(TGTGAb.sup.6 GCTGTG)-3' 13
110 75 ______________________________________ .sup.a Data
previously published (Crone et al. (1993), supra; and Crone e al.
(1994), supra). .sup.b Data previously published (Moschel et al.
(1992), supra).
The results demonstrate that a single-stranded
oligodeoxyribonucleotide comprising O.sup.6 -benzylguanine (b.sup.6
G), even one as short as three nucleotides, was more effective in
inactivating wild-type and mutant AGTs than the free base O.sup.6
-benzylguanine or O.sup.6 -benzyl-2'-deoxyguanosine. In this
regard, oligodeoxyribonucleotides, which were from 5 to 11
nucleotides in length and which comprised O.sup.6 -benzylguanine,
inactivated the mutant AGTs P140A and G156A at 4-fold and 11-fold
higher concentrations, respectively, than the concentration
required to inactivate wild-type AGT, whereas O.sup.6
-benzylguanine inactivated the mutant AGTs P140A and G156A at
25-fold and 300-fold higher concentrations, respectively, than the
concentration required to inactivate wild-type AGT. Maximal
effectiveness was observed for oligodeoxyribonucleotides that were
from about 5 to about 11 nucleotides in length. There was no
significant loss (<5%) of AGT activity in the absence of
inactivator.
All of the references cited herein, whether patents, patent
applications or publications, are hereby incorporated in their
entireties by reference.
While this invention has been described with an emphasis upon
preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations of the preferred embodiments can
be used and that it is intended that the invention can be practiced
otherwise than as specifically described herein. Accordingly, this
invention includes all modifications encompassed within the spirit
and scope of the invention as defined by the following claims.
__________________________________________________________________________
# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii)
NUMBER OF SEQUENCES: 1 - - - - (2) INFORMATION FOR SEQ ID NO:1: - -
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 base - #pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear -
- (ii) MOLECULE TYPE: DNA (synthetic) - - (iii) HYPOTHETICAL: NO -
- (iv) ANTI-SENSE: NO - - (ix) FEATURE: (A) NAME/KEY: misc.sub.-- -
#feature (B) LOCATION: 6 (D) OTHER INFORMATION: - #The guanine at
position 6 is O6-benzylgua - #nine - - (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:1: - - TGTGAGCTGT G - # - # - # 11
__________________________________________________________________________
* * * * *